The goals of the proposed studies are to conduct a thorough preclinical evaluation of promising HIV resistance genes in primary CD4+ lymphocytes, and to establish appropriate procedures and necessary logistical support for clinical studies targeting peripheral T cells. We haven previously shown that expression of TAR decoy RNA in immortalized CD4+ T-cells provided significant protection against HIV replication, and initial studies have indicated that transduction of primary CD4+ PBL obtained from healthy volunteers with TAR decoy vectors led to a modest though reproducible inhibition of HIV replication. We have recently identified a highly potent RRE-decoy comprising of the 13 nt-long minimal Rev binding domain of the HIV-1 RRE which unlike TAR decoy RNA is not known to bind cellular factors that could be sequestered in uninfected cells expressing decoy RNA, and potentially compromise the viability or function of the genetically modified cells. The specific objectives of the proposed studies are: (a) To develop increasingly potent inhibition strategies based on RRE-decoys and combination vectors including TAR decoys, and rev transdominants and ribozymes. (b) To evaluate inhibition strategies in ex vivo cultured CD4+ PBL obtained from healthy volunteers and HIV infected individuals including the development of methods to maximize efficiency of retroviral gene transfer, comparative evaluation of HIV resistance genes against clinical HIV isolates, and the development of highly accurate and sensitive PCR-based methods to measure gene transfer and HIV replication in preclinical models and clinical settings. (c) To establish procedures and carry out careful and exhaustive safety studies as mandated by the FDA, in ex vivo cultured CD4+ PBL and in hematochimeric SCID mice reconstituted with CD4+ PBL obtained from HIV infected individuals. The studies proposed in this application will set the stage for clinical evaluation of promising HIV resistance genes in a peripheral T-cell- targeted gene therapy protocol, and will provide the basis for clinical studies targeting hemopoietic stem cells.